Tools for high-throughput nucleic acid analysis are becoming increasingly important in light of recent advancements in availability of nucleic acid sequence information and genomic data for humans and other organisms. The techniques of ligation-mediated detection of nucleic acids, coupled with hybridization of nucleic acids on arrays are widely used form the basis of genomics applications such as oligonucleotide ligation assay (OLA) (Landegren et al., Science, 241:1077 (1998)), ligase chain reaction (Barany, Proc. Natl. Acad. Sci. USA, 88:189 (1991)), and ligation of padlock or open circle probes (Nilsson et al., Science, 265:2085 (1994)). Ligation-mediated nucleic acid detection methodology relies on the ability of ligases to accurately discriminate between highly homologous nucleotide sequences, differing in some instances only at the terminal nucleotide position. Thus far, most ligation applications involve DNA-templated DNA ligation by a DNA ligase, where both the target nucleic acid and the oligonucleotide probes consist of deoxyribonucleotide polymers. Such applications are particularly useful for generating genomic sequence data and SNP profiling.
RNA-templated DNA ligation is an attractive method for detection of RNA, determination of RNA sequence identity, expression monitoring and transcript analysis. Direct detection of RNA target-DNA probe duplexes (without first converting RNA to cDNA by reverse transcription) has been challenging because a majority of tested DNA ligases fail to ligate nicked DNA on an RNA template. The exception to this is T4 DNA ligase, which is able to ligate nicked DNA hybridized to a RNA strand at a depressed rate.
T4 DNA ligase is an enzyme belonging to the DNA ligase family of enzymes (E.C. 6.5.1.1) which catalyzes the formation of a covalent phosphodiester bond from a free 3′ hydroxyl group on one DNA molecule and a free 5′ phosphate group of a second, separate DNA molecule, thus covalently linking the two DNA strands together to form a single DNA strand. This activity may also be applied to RNA and is especially useful in molecular genetics where sticky (or blunt) ends of double-stranded DNA (dsDNA) may be fused together with other dsDNA molecules, both products of a restriction enzyme cut, for instance. DNA ligases play critical roles in cell division, in a process called lagging strand DNA replication, as well as cell recovery in the dsDNA break repair mechanism. DNA ligases also play critical roles in normal cellular processes used to generate diversity in the immune system pathways, i.e. during V(D)J recombination. Commercially exploited DNA ligases include the bacteriophage T4 DNA ligase. T4 DNA ligase possesses the basic activity of catalyzing formation of covalent phosphodiester bonds, as described above, but only operates on double-stranded molecules, i.e. DNA/DNA, DNA/RNA hybrids, and RNA/RNA. Like many ligases, T4 DNA ligase activity requires adenosine triphosphate (ATP) as a cofactor. Recombinant T4 DNA ligase, and various mutants thereof, is commercially available.
The ligation reaction catalyzed by DNA ligase occurs in three general steps. First, the ligase enzyme is activated by charging with ATP. Addition of ATP to ligase enzyme causes formation of an intermediate AMP-enzyme species concomitant with hydrolysis of ATP to yield AMP. Second, the charged AMP-enzyme intermediate binds to the dsDNA (or dsRNA, or RNA/DNA complex) and transfers the AMP moiety to the free 5′ terminal phosphate, to form a high energy 5′-5′ phosphate bond. Third, the enzyme provides the appropriate environment in which the 3′ hydroxyl group of the second strand of DNA (or RNA) is able to attach the high energy 5′-5′ phosphate bond, thereby forming a covalent phosphodiester bond as a product and releasing ligase enzyme and AMP. Free enzyme does not bind the intermediate high energy 5′-5′ phosphate bond species to an appreciable amount. Thus, if the ligase prematurely releases from the duplex after formation of the high energy 5′-5′ phosphate bond, the reaction will typically end and the intermediate will not proceed to the final ligated product.
Methods are disclosed herein that provide for efficient RNA-templated DNA ligation.